A cosmid for selecting genes by complementation in Aspergillus nidulans : Selection of the developmentally regulated yA locus

Abstract

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in Aspergillus nidulans. pKBY2 contains the bacteriophage X cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC' gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, Bgl II, Mbo I, or Sau3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35-to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA ) pabaA-tpC-Aspergillus strain (FGSC237) to tipC' at frequencies of approximately 10 transformants per pg of DNA. Three of approximately 1000 tipC' pabaA-colonies obtained were putative yA+ transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced E. coli to ampicillin resistance following treatment in vitro with a X packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded tipC' transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.Complementation of mutations in Saccharomyces cerevisiae (yeast) and in animal cells by transformation with recombinant DNA libraries provides a convenient and valuable method for the isolation of specific eukaryotic genes (1-4). Plasmid "shuttle" vectors for this purpose typically include genes for selection in both Escherichia coli and the eukaryotic host and one or more unique restriction sites for insertion of foreign DNA fragments. In addition, these vectors must transform the eukaryotic host at a frequency sufficient to allow screening of entire genomes and should provide a simple way to recover the complementing DNA in the prokaryotic host. Shuttle vectors for S. cerevisiae frequently contain sequences mediating autonomous replication in yeast to enhance the frequency of transformation and to allow recovery of plasmids by direct transformation of E. coli with total DNA isolated from yeast cells (5-9). Generally, analogous vectors have been constructed for use with cultured mammalian cells (10, 11). However, because of restrictions in the size of DNA fragments that can be introduced into these plasmids, it is difficult to screen entire animal genomes. Some shuttle vectors for mammalian cells have been constructed that contain one or two bacteriophage X cos sites to allow in vitro assembly of phage particles containing large (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) kilobase) cloned inserts, in order to reduce this problem (3, 4).Aspergillus nidulans is an Ascomycetous fungus related to S. cerevisiae and,...

Lignin peroxidase gene family of Phanerochaete chrysosporium: complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome

“…Cosmid pQQ24 is a pWE15 derivative (35) containing LiP genes LiPA, LiPB, and GLG5 (9). Single LiP genes are contained in cosmid pKBY2 (38) derivatives and in pO282 and pV4 (30). Subclone pO282::910 contains a 910-bp XhoI fragment of 0282 in Bluescript KSII (Stratagene Inc., La Jolla, Calif.).…”

RecA-independent high-frequency deletion of recombinant cosmid DNA in Escherichia coli

“…deletions in cosmid DNAs during their amplification in Escherichia coli host cells : probably because of their large size, cosmids do not seem to be stably maintained in E. coli hosts. These deletions have been mentioned in several papers (Chia et al, 1982;Steinmetz et al, 1982;Yelton et al, 1985), but they have not yet been extensively characterized, and the primary importance of recA mutations in E. coli hosts to prevent the cosmid deletion events has been presumed rather than investigated systematically (Ish-Horowicz & Burke, 1981 ;Chia et al, 1982;Maniatis et al, 1982). Several mechanisms have been proposed for the various deletion events that occur in recA E. coli hosts (Brutlag et al, 1977;Collins, 1980;Lilley, 1981 ;Liu &Lark, 1982;Collins et al, 1982;Boissy & Astell, 1985;Saing et al, 1988).…”

Regulation of the Aspergillus nidulans Pectate Lyase Gene (pelA)

“…trpCz~B is a functional allele in which the single BamHI site in the trpC locus has been eliminated by in vitro mutagenesis (Yelton et al, 1985). The PL cDNA clone pRD091 was used to isolate pL30C5 from an ordered Lorist2 (Gibson et al, 1987) Miller et al, 1985).…”

“…The polarity and approximate position of the pelA transcription unit shown in Figure 5A was determined from the restriction map of pRD091. Plasmid pRD303 was constructed by replacing a 1.4-kb Sail fragment containing the 3' half of pelA with a fragment containing the A. nidulans frpCAS gene ( Figure 5A; see Yelton et al, 1985). The 8.3-kb BamHI fragment was isolated from the plasmid and used to transform a frpC" A. nidulans strain (FGSC237) with PL antiserum.…”